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Biochemistry Lab Report Part_B

The document outlines a biochemistry lab experiment focused on varying the amount of enzyme in a reaction, specifically investigating turnip peroxidase activity. It includes data tables, a discussion on controls, and methods to confirm the enzyme's identity and activity levels. Key findings indicate that a 200 μl volume of turnip extract yields a linear curve, with saturation of active sites occurring only at specific concentrations.

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33 views

Biochemistry Lab Report Part_B

The document outlines a biochemistry lab experiment focused on varying the amount of enzyme in a reaction, specifically investigating turnip peroxidase activity. It includes data tables, a discussion on controls, and methods to confirm the enzyme's identity and activity levels. Key findings indicate that a 200 μl volume of turnip extract yields a linear curve, with saturation of active sites occurring only at specific concentrations.

Uploaded by

Iva
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
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Biochemistry Lab II

University of Houston BCHS 4311

Lab report

Part B. Varying the Amount of Enzyme in the Reaction

Table 1. Reagents required for part B. Notice that the total volume for each tube is 3 ml.
Table 2. Absorbance readings for investigating the effect of varying the amount of enzyme

in the reaction.

Table 3. Absorbance readings over time at increasing enzyme concentrations.


Data:

1. Plot a graph for varying the amount of enzyme (absorbance vs time)

(Table 3).

Discussion for Part B:

1. Explain the control in this experiment (the what and why).

In this experiment, H2O2 (the substrate) wasn’t in tube 1 (the blank) but the turnip extract,

therefore, tube 1 was utilized as the control. If no reaction is occurring in the control test

tube the Spectronic 200 can be adjusted. The presence of hydrogen peroxide would

cause the appearance of a reaction.


2. Can you be absolutely sure that you are testing peroxidase? What would you

need to do to be sure turnip peroxidase is responsible for the color change

of guaiacol and not some other turnip enzyme?

No, I can’t be sure that I am testing peroxidase. When hydrogen peroxide is decreased

by the turnip peroxidase a tetraguaiacol is formed and guaiacol is oxidized. The

production of tetraguaiacol can be estimated by following the absorbance at a wavelength

of 470 nm.

3. What volume of turnip extract (or range of volumes) yielded a linear curve

for your turnip peroxidase activity?

A 200 μl volume of turnip extract yielded a linear curve for the turnip peroxidase activity.

4. What volume of turnip extract will you use for the remaining sections of this

lab exercise and why did you select this volume?

I will use a range of volume from 60 to 600 μl of turnip extract for the remaining sections

of the lab exercise because I want to find a volume that produces a linear plot over at

least a two-minute time course.

5. Does the reaction go to completion (absorbance reaches a maximum

threshold) for any of the enzyme concentrations tested? If so, which

concentrations?

The reaction goes to completion (absorbance reaches a maximum threshold) for 180 μl

concentration of the enzyme tested.


6. Are all of the turnip peroxidase active sites saturated in any of the reactions?

Which ones and how do you know?

The turnip peroxidase active sites are unsaturated in reactions 1-9. This is known

because the rate of reaction increases. All of the turnip peroxidase active sites are

saturated only in reaction number 10 because the reaction rate starts decreasing.

7. How might you determine how much peroxidase is ACTUALLY in the volume

of extract used to create your linear curve? Think about the experiments

you might need to do. Refer to your biochemistry textbook if you need ideas.

I will determine the concentration of peroxidase in the volume of the extract used to create

a linear curve by the following steps:

 Determine the amount of substrate needed to achieve maximum saturation for the

peroxidase by including certain amounts of substrate and straight away measuring

equilibrium after inclusion, and

 Mutate the substrate concentration until an appropriate concentration is

determined.

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